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Introduction

Background

The liver is the major organ for metabolism of endogenous substrates as well as exogenous drugs. There are several in vitro tools available to help researchers study the metabolic fate of drug candidates, including isolated fresh or cryopreserved hepatocytes, liver slices, and sub-cellular fractions such as liver microsomes and S9 fractions. These sub-cellular fractions are prepared from the liver via a series of homogenization and ultracentrifugation steps.

An initial lower speed centrifugation of liver homogenate at 10,000g produces the S9 fraction also known as the supernatant of this centrifugation. The S9 fraction contains all phase I and phase II enzymes. A further centrifugation of the S9 fraction at 100,000g yields the endoplasmic reticulum-derived microsomes. Microsomes are an enriched source of cytochrome P450 (CYP) and flavin monooxygenases (FMO) enzymes. Additionally, some phase II enzymes (e.g. certain uridine glucuronide transferases (UGT) isoforms and epoxide hydrolase (EH) enzymes) are present in microsomes. . Microsomes can be used to investigate UGT activity; however, microsomal membranes restrict access of UGT substrates and/or cofactors. Optimal UGT activity can be achieve by the addition of MgCl2 and a pore-forming antibiotic (i.e. alamethicin). These components allow for the efficient transfer of a glucuronide product and the co-factor, uridine 5’-diphospho-alpha-D-glucuronic acid (UDPGA) within the microsomal matrix. Individual or pooled donor microsomes can be used for metabolism-related studies. Pooled donors can represent the “average” human population or particular factors of research interest, such as age, BMI, or limited capabilities for certain CYP isoforms.

Important notes

The following is a general procedure for metabolism studies in liver microsomes. It is recommended that the metabolism of a test article be measured under initial rate conditions. Since every test article is different, each will require separate optimization of microsomal protein concentration, test article concentration, and incubation times. Ideally, the amount of substrate consumed during the reaction should be 10% to 15% in order to measure initial rates of metabolite formation.

Microsomes should be stored at -80°C until immediately prior to experiment. Microsomes can be refrozen twice without compromising enzymatic activity.